The invention relates to new antithrombotic agents, a process for their preparation, pharmaceutical compositions containing the compounds as active ingredients, as well as the use of said compounds for the manufacture of medicaments.
Serine proteases are enzymes which play an important role in the blood coagulation cascade. Members of this group of proteases are for example thrombin, trypsin, factors VIIa, IXa, Xa, XIa, XIIa, and protein C. Thrombin is the final serine protease enzyme in the coagulation cascade. The prime function of thrombin is the cleavage of fibrinogen to generate fibrin monomers, which are cross-linked to form an insoluble gel. In addition, thrombin regulates its own production by activation of factors V and VIII earlier in the cascade. It also has important actions at cellular level, where it acts on specific receptors to cause platelet aggregation, endothelial cell activation and fibroblast proliferation. Thus thrombin has a central regulatory role in haemostasis and thrombus formation. Since inhibitors of thrombin may have a wide range of therapeutical applications, extensive research is done in this area. Another important serine protease, factor Xa, catalyzes the conversion of prothrombin into thrombin.
In the development of synthetic inhibitors of serine proteases, and more specifically of thrombin, the benzamidine moiety is one of the key structures. It mimics the protonated side-chain of the basic amino acids Arg and Lys of its natural substrates. Compounds with this moiety have been studied extensively and repeatedly. A very potent representative of this type of thrombin inhibitors is the amino acid derivative Nxcex1-(2-naphthylsulfonyl)-glycyl-4-amidinophenylaianinpiperidide (NAPAP) (Stxc3xcrzebecher, J. et al., Thromb. Res. 29, 635-642, 1983). However, the profile of NAPAP is unattractive for therapeutical applications: for example, the compound has low thrombin specificity and is poorly soluble. Derivatives of NAPAP were subsequently prepared, such as the N-alkyl substituted derivatives disclosed in EP 0,236,163 or the glycopeptide derivatives described EP 0,558,961, Proc. Am. Pept. Symp., 13th (60LXAW); 94; pp. 643-5 (Stxc3xcber, W. et al., Pept.: Chem., Struct. Biol.,), Proc. Int. Symp. Controlled Release Bioact. Mater. (PCRMEY, 10220178); 94;Vol. 21 st; pp. 712-12 (Walter, E. et al.), and EP 0,513,543. However, although these derivatizations may have led to improvements of the pharmacological profile when compared to NAPAP, all such NAPAP-derived compounds are still active only as direct thrombin inhibitors and they have a restricted plasma half-life and a fast clearance (thus displaying their anti-thrombin activity only for a short period of time).
It has now been found that compounds of the formula (I) 
wherein
R1 is phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, (iso)quinolinyl, tetrahydro(iso)quinolinyl, 3,4-dihydro-1H-isoquinolinyl, chromanyl or the camphor group, which groups may optionally be substituted with one or more substituents selected from (1-8C)alkyl or (1-8C)alkoxy;
R2 and R3 are independently H or (1-8C)alkyl;
R4 is (1-8C)alkyl or (3-8C)cycloalkyl;
or R3 and R4 together with the nitrogen atom to which they are bonded are a nonaromatic (4-8)membered ring optionally containing another heteroatom, the ring optionally being substituted with (1-8C)alkyl or SO2-(1-8C)alkyl;
Q is a spacer having a chain length of 10 to 70 atoms; and
Z is a negatively charged oligosaccharide residue comprising two to six mionosaccharide units, the charge being compensated by positively charged counterions;
or a pharmaceutically acceptable salt thereof or a prodrug thereof are potent and highly versatile antitbrombotics. The compounds of the invention have antithrombin activity, but also the structure of the compounds may be selectively modified so that they have a tuneable mixed profile of both non-mediated, direct anti-thrombin (factor IIa) activity And antithrombin III (AT-III) mediated anti-Xa activity. The compounds of the invention thus are dual inhibitors. Compounds of the invention have a long plasma half-life and, as a result, they possess prolonged anti-thrombin activity compared to NAPAP or its above reported derivatives. Further, compounds of the invention may escape the neutralizing action of platelet factor 4 (PF4). Low toxicity is also an advantageous aspect of compounds of this invention.
Another type of dual inhibitors is disclosed in EP 0,649,854. Contrary to the compounds of the present invention the conjugated saccharide compounds disclosed in that document display indirect, AT-III mediated anti-thrombin activity, in addition to AT-III mediated anti-Xa activity.
The compounds of the present invention are useful for treating and preventing thrombin-mediated and thrombin-associated diseases. This includes a number of thrombotic and prothrombotic states in which the coagulation cascade is activated which include, but are not limited to, deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterial occlusion from thrombosis or embolism, arterial reocclusion during or after angioplasty or thrombolysis, restenosis following arterial injury or invasive cardiological procedures, postoperative venous thrombosis or embolism, acute or chronic atherosclerosis, stroke, myocardial infarction, cancer and metastasis, and neurodegenerative diseases. The compounds of the invention may also be used as anticoagulants in extracorporeal blood circuits, as necessary in dialysis and surgery. The compounds of the invention may also be used as in vitro anticoagulants.
The mixed profile of the compounds of the invention may be tuned by varying the nature of the oligosaccharide residue Z and the length of the spacer Q. A range of profiles is thereby available.
Any negatively charged oligosaccharide residue of 2 to 6 saccharide units is useable in the compounds of the present invention. Suitable compounds of the invention are compounds wherein Z is a sulfated or phosphorylated oligosaccharide residue. Preferably, the oligosaccharide residue Z is derived from an oligosaccharide which has per se AT-III mediated anti-Xa activity, such as the saccharides disclosed in EP 0,454,220 and EP 0,529,715. Particularly preferred oligosaccharide residues are pentasaccharide residues. Most preferably, Z has the formula (II) 
wherein R5 is independently OSO3xe2x88x92 or (1-8C)alkoxy.
Further preferred compounds of the invention are compounds of formula I, wherein R1 is phenyl, 4-methoxy-2,3,6-trimethylphenyl or naphthyl. In preferred compounds, NR3R4 represents the piperidinyl group. Preferably, R2 is H.
The chemical structure of the spacer is of minor or no importance for the anti-thrombotic activity of the compounds of the invention, it may however not be completely rigid. Highly flexible spacers are more suitable than others.
Further, for synthetic reasons some spacers are more appropriate than others. Suitable spacers that can easily be used have for example the formula (III):
xe2x80x94[(CH2)2O]mxe2x80x94[(CH2)nxe2x80x94NR3xe2x80x94C(O)]pxe2x80x94Wxe2x80x94(CH2)sxe2x80x94xe2x80x83xe2x80x83(III),
wherein
W
is xe2x80x94[1,4-phenylene-NR3xe2x80x94C(O)]qxe2x80x94(CH2)rxe2x80x94Sxe2x80x94 or
xe2x80x94(CH2)txe2x80x94Sxe2x80x94(CH2)uxe2x80x94[O(CH2)2]vxe2x80x94Oxe2x80x94(CH2)wxe2x80x94C(O)xe2x80x94NR3xe2x80x94;
and R3 is independently H or (1-8C)alkyl;
m=1-12; n=1-8; p=0-4; q=0 or 1; r=1-8; s=1-8; t=1-8; u=1-8; v=1-12; w=1xe2x88x9d8; the total number of atoms is 10-70; and the moiety xe2x80x94[(CH2)2O]mxe2x80x94 is the end with which Q is attached to Z.
Preferred spacers are the following:
xe2x80x94[(CH2)2O]5xe2x80x94(CH2)2xe2x80x94NHxe2x80x94C(O)xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94;
xe2x80x94[(CH2)2O]5xe2x80x94(CH2)2xe2x80x94NHxe2x80x94C(O)xe2x80x94CH2xe2x80x94Sxe2x80x94(CH2)2xe2x80x94[O(CH2)2]3xe2x80x94Oxe2x80x94CH2xe2x80x94C(O)xe2x80x94NHxe2x80x94(CH2)4xe2x80x94; and
xe2x80x94[(CH2)2O]3xe2x80x94(CH2)2xe2x80x94NHxe2x80x94C(O)-1,4-phenylene-NHxe2x80x94C(O)xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94.
In the description of the compounds of formula (I) the following definitions are used.
The term (1-8C)alkyl means a branched or unbranched alkyl group having 1-8 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, hexyl and octyl. Methyl and ethyl are preferred alkyl groups.
The term (1-8C)alkoxy means an alkoxy group having 1-8 carbon atoms, the alkyl moiety having the meaning as previously defined. Methoxy is a preferred alkoxy group.
The term (3-8C)cycloalkyl means a cycloalkyl group having 3-8 carbon atoms, being cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclo-octyl. Cyclopentyl and cyclohexyl are preferred cycloalkyl groups.
The spacer length is the number of atoms of the spacer, counted along the shortest chain between Z and the peptide part of the molecule, not counting the oxygen atom of the oligosaccharide Z which is connected to the spacer.
The term xe2x80x9cprodrugxe2x80x9d means a compound of the invention in which the amino group of the amidino-moiety is protected, e.g. by hydroxy or a (1-6C)alkoxycarbonyl group.
The compounds of the present invention are prepared by derivatizing NAPAP (or a NAPAP-analogue) at the glycine position with cysteine or lysine using methods generally known in the art, which compound subsequently (a) is coupled to a oligosaccharide-spacer residue or (b) is coupled to a spacer, which then is derivatized with a thiol group and subsequently is coupled to an oligosaccharide residue. Any suitable oligosaccharide may be used for this purpose, for example oligosaccharides known in literature (e.g. from EP 0,454,220 and EP 0,529,715, but not limited to these sources) or commercially available oligosaccharides. The oligosaccharides may be phosphorylated at an appropriate time by methods e.g. described by Buijsman, R. et al. (Abstracts of papers, 9th European Carbohydrate Symposium Utrecht 1997, Abstract A150). The coupling of the spacer to the oligosaccharide can for instance be performed by using the methods described in EP 0,649,854.
The peptide coupling, a procedural step in the above described method to prepare the compounds of the invention, can be carried out by methods commonly known in the art for the couplingxe2x80x94or condensationxe2x80x94of peptide fragments such as by the azide method, mixed anhydride method, activated ester method, or, preferably, by the carbodiimide method, especially with the addition of catalytic and racemisation suppressing compounds like N-hydroxysuccinimide and N-hydroxybenzotriazole. An overview is given in The Peptides, Analysis, Synthesis, Biology, Vol 3, E. Gross and J. Meienhofer, eds. (Academic Press, New York, 1981).
Amine functions present in the compounds may be protected during the synthetic procedure by an N-protecting group, which means a group commonly used in peptide chemistry for the protection of an xcex1-amino group, like the tert-butyloxycarbonyl (Boc) group, the benzyloxycarbonyl (Z) group, the 9-fluorenylmethyloxycarbonyl (Fmoc) group or the phthaloyl (Phth) group. Removal of the protecting groups can take place in different ways, depending on the nature of those protecting groups. Usually deprotection takes place under acidic conditions and in the presence of scavengers. An overview of amino protecting groups and methods for their removal is given in the above mentioned The Peptides, Analysis, Synthesis, Biology, Vol 3.
The compounds of the invention, which can occur in the form of a free base, may be isolated from the reaction mixture in the form of a pharmaceutically acceptable salt. The pharmaceutically acceptable salts may also be obtained by treating the free base of formula (I) with an organic or inorganic acid such as HCl, HBr, HI, H2SO4, H3PO4, acetic acid, propionic acid, glycolic acid, maleic acid, malonic acid, methanesulphonic acid, fumaric acid, succinic acid, tartaric acid, citric acid, benzoic acid, ascorbic acid and the like.
The compounds of this invention possess chiral carbon atoms, and may therefore be obtained as a pure enantiomer, or as a mixture of enantiomers, or as a mixture containing diastereomers. Methods for obtaining the pure enantiomers are well known in the art, e.g. crystallization of salts which are obtained from optically active acids and the racemic mixture, or chromatography using chiral columns. For diastereomers straight phase or reversed phase columns may be used.
The compounds of the invention may be administered enterally or parenterally. The exact dose and regimen of these compounds and compositions thereof will neccessarily be dependent upon the needs of the individual subject to whom the medicament is being administered, the degree of affliction or need and the judgment of the medical practitioner. In general parenteral administration requires lower dosages than other methods of administration which are more dependent upon absorption. However, the daily dosages are for humans preferably 0.001-100 mg per kg body weight, more preferably 0.01-10 mg per kg body weight. The medicament manufactured with the compounds of this invention may also be used as adjuvant in acute anticoagulant therapy. In such a case, the medicament is administered with other compounds useful in treating such disease states. Mixed with pharmaceutically suitable auxiliaries, e.g. as described in the standard reference, Gennaro et al., Remington""s Pharmaceutical Sciences, (18th ed., Mack Publishing Company, 1990, see especially Part 8: Pharmaceutical Preparations and Their Manufacture) the compounds may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories. By means of pharmaceutically suitable liquids the compounds can also be applied in the form of a solution, suspension, emulsion, e.g. for use as an injection preparation, or as a spray, e.g. for use as a nasal spray.
For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like is contemplated. In general any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used. Suitable carriers with which the compositions can be administered include lactose, starch, cellulose derivatives and the like, or mixtures thereof, used in suitable amounts.